The present invention relates to novel anthracyclines generated by cloning of fused genes in a mutated Streptomyces galilaeus strain. The aglycone moieties of the compounds obtained are modified aklavinones, the modifications being caused by the genes introduced into the mutant, and the sugar moieties are those derived from the host strain.
Anthracyclines, which are mainly produced by Streptomyces species as secondary metabolites, share a great value as antitumor agents. Generally, the anthracycline molecule comprises an aglycone backbone and one or more sugar moieties attached therein. Aclacinomycins are a group of anthracyclines sharing aklavinone as the aglycone moiety and a sugar residue, as rhodosamine (Aclacinomycin T), wherein 2-deoxyfucose (Aclacinomycin S) and a third sugar, originally rhodinose, can be sequentially attached. Modifications of the third sugar residue are common. The first aclacinomycin complex consisting of Aclacinomycin A (AcmA), B (AcmB), and Y (AcmY) was first isolated from S. galilaeus by Oki et al., (1975). Later, aclacinomycin A, Aclarubicin, was passed through clinical trials for use in cancer chemotherapy. Research to obtain aclacinomycins with minor modifications has been carried out to find new candidates for drug development. During these studies compounds produced by the mutated S. galilaeus strains possessing modifications in the sugar moiety have been found (Matsuzawa et al., 1981).
The general formula of aclacinomycins (Acm) comprising aklavinone and an aminosugar, rhodosamine (Rhn) is as follows 
wherein R is H or a sugar residue being a mono- or a disugar-moiety.
In addition, aklavinone glycosides which have no aminosugar moieties have been produced by S. galilaeus strains obtained by mutagenesis treatment (Matsuzawa et al., 1981 and Ylihonko et al., 1994).
Aclacinomycin A, called Aclarubicin, is a unique member of aclacinomycin group used in cancer chemotheraphy. It is mainly used for leukemia. Doxorubicin, an anthracycline representing the daunomycin group, is the most widely used cytostatic antibiotic, showing activity against quite a variety of tumors. Unfortunately, the harmful side-effects such as cumulative cardiotoxicity cause the treatment by doxorubicin to be aborted despite of its effectiveness. Instead, cardiotoxicity is not a common side-effect in aclarubicin treatment. Nevertheless, the lack of activity on solid tumors limits its use mainly to leukemia and lymphomas. For this reason an optimal anthracycline for cancer treatment would possess clinical applicability similar to that of doxorubicin, whereas side-effects should rather be as those of aclarubicin, in accordance with its mode of action. Current status of cancer chemotheraphy perpetuates the need of novel molecules in order to develop new cytostatic drugs.
Matsuzawa et al. (1981) describe production of aklavinone-rhodosamine-2-deoxy-fucose-2-deoxyfucose. This aclacinomycin has been isolated from S. galilaeus mutant strains 7U-491 and 7N-1881 derived from the strain S. galilaeus ATCC 31133. As well, the strains 7U-491 and 7N-1881 produced aklavinone-Rhn-dF and, in addition, aklavinone-dF-dF (MA144 U9) has been obtained from the culture of 7N-1881.
We mutated a Streptomyces galilaeus wild type strain (ATCC 31615) in order to make the mutant to produce a modified anthracycline. The S. galilaeus mutant obtained was named as H075, and deposited under the rules of the Budapest Treaty on Jun. 30, 1997 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, Braunschweig, Germany with the accession number DSM 11638. S. galilaeus H075 itself produces aklavinone-rhodosamine-2-deoxyfucose-2-deoxyfucose (H075-1a) as the major product, and minor amounts of aklavinone-dF-dF-dF (H075-1d), an anthracycline which has no aminoglycosides. H075 thereby differs from the producer strains of Matsuzawa et al. (1981), since a novel compound, aklavinone-deoxyfucose-deoxyfucose-deoxyfucose is produced as well. This mutant strain was used as the host strain to express the known genes for nogalamycin (sno) and rhodomycin (rdm) biosynthesis to produce novel hybrid anthracyclines. The sno1-3 genes derived from S. nogalater cause a replacement of an ethyl group at C-9 to a methyl group of aklavinone. The rdmB and E genes derived from S. purpurascens are responsible for hydroxylation of the positions 10 and 11, respectively, and rdmC gene product decarboxylates the position 10 in the aglycone moiety. This results in novel compounds useful in cancer chemotherapy.
A specific object of this invention is thus a process for producing new hybrid anthracyclines by transforming the genes for nogalamycin (sno) and/or rhodomycin (rdm) biosynthesis to a S. galilaeus strain, preferably to the H075 mutant.
The compounds produced by expressing said genes in S. galilaeus H075 to change the substituents at positions 9, 10 and 11 in aklavinone are included in this invention.
The S. galilaeus mutant H075 (DSM 11638), which is used as the host for production of novel anthracyclines according to this invention is an overproducer of aklavinone glycosides. The general formula II for the said compounds is 
wherein
R1=CH2CH3 or CH3,
R2=COOCH3, H or OH and
R3=OH or H.
In the following description the compounds are designated according to formula II above in the manner that a number is given to each aglycone based on the R-groups listed above, whereas small letters refer to sugar moieties according to formula II, a=Rhn-dF-dF; b=Rhn-dF; c=Rhn; d=dF-dF-dF; e=dF-dF. (Rhn=rhodosamine, dF=deoxyfucose).
The transformed strains obtained using S. galilaeus H075 as a host produce altogether 5 (sugar moiety)xc3x9711 (aglycone modifications) different anthracyclines.
The invention concerns specifically the new compounds H075-2a,b,c,d,e, H075-3a,d,e, H075-4a,b,c,d,e, H075-5a,b,c,d,e, H075-6a,b,c,d,e, H075-7a,b,c,d,e, H075-8a,b,c,d,e, H075-9a,b,c,d,e, H075-10a,d,e, H075-11a,b,c,d,e, H075-12a,b,c,d,e as given above in Table 1, and in specific the novel compounds H075-2a, -2b and -2d; H075-3d; H075-4a; H075-5e; H075-6b and -6c; H075-8a; H075-10a; and H075-11b and -11e, as defined in claim 1.
The prior known compounds related to the compounds of the present inventionxe2x80x94as named according to the formula IIxe2x80x94are:
H075-1a (MA144 U1, Matsuzawa et al., 1981),
H075-1b (AcmS, Oki et al., 1977),
H075-1c (Aklavin, AcmT, Streliz et al., 1956),
H075-1e (MA144 U9, Matsuzawa et al., 1981),
H075-3b (Auramycin C, Hoshino et al., 1982),
H075-3c (Auramycin D, Hoshino et al., 1982),
H075-10b (Betaclamycin S, Yoshimoto et al., 1984) and
H075-10c (Betaclamycin T, Yoshimoto et al., 1984).
The present invention enables the drug development of compounds of the formula II. According to the invention the compounds of the present study are active against the MES-SA cell line as compared with daunomycin and aclarubicin. As well, in vivo activity using P388 leukemia cells in mice was comparable with that of daunomycin. The tumor cell panel used for investigation on in vitro cytostatic activity suggests the properties relative to aclacinomycins or daunomycins depending on the substituents of the aglycone moiety. The results are encouraging to find novel pharmaceuticals for cancer treatment.
The compounds produced by the transformed strains are more hydrophilic than aclarubicin due to their chemical structures. For this reason, they resemble doxorubicin more than aclarubicin in their physicochemical properties. Doxorubicin acts on tumor cells with a mechanism distinct from that of aclarubicin. It is suggested to be an inhibitor of topoisomerase II stabilizing a drug-DNA-enzyme complex to prevent a ligation of DNA strands in double helix. This results in cell cycle arrest at phase G2. Aclarubicin is supposed to inhibit the interaction of topoisomerases with DNA, although the mechanism is not well known and the compound is found to cause the cell cycle to arrest at phase G1. The hybrid anthracyclines of the present invention were found to arrest the cell cycle at phase G1, the commitment point of chromosome replication, showing thus a mode of action similar to that of aclarubicin.
The compounds of this invention could be produced in any microbial cell able to express the genes for anthracyclines. Streptomyces species are advantageous for the purposes of this invention, and a specific embodiment comprises the use of the genetically engineered S. galilaeus H075 (DSM 11638) obtained by mutagenization of S. galilaeus ATCC 31615 wild type by NTG (N-methyl-Nxe2x80x2-nitro-N-nitroso-guanidine).
In Streptomyces the biosynthesis of aklavinone proceeds via the polyketide pathway. Polyketide synthase (PKS) is responsible for the formation of the polyketide backbone. Three specific gene products constituting the xe2x80x9cminimal PKSxe2x80x9d do control the acceptance of the starter unit as well as the number of the extender units (acetate) used in the polyketide assembly. The starter unit for the aglycone biosynthesis determines the substituent at position 9 in the structure shown above. It is previously known that propionate is used as the starter in aklavinone biosynthesis wherein R1 is an ethyl group. This group could be converted to a methyl group by expressing the minimal PKS genes for nogalamycin in H075, as acetate is used to start the biosynthesis pathway for nogalamycin (Ylihonko et al., 1996). The genes for nogalamycin minimal PKS (sno1-3) can be isolated from the plasmid pSY15 (DSM 9436) as a 5.4 kb DNA-fragment restricted with SacI-BglII to give pSY21. The fragment is cloned in a multicopy plasmid vector replicating in Streptomyces species.
The substituent at position 10 in aklavinone (group R2) is a carbomethoxy group. This group is removed by the activity of an esterase, preferably by RdmC to produce the compounds H075-7, H075-8, H075-9 and H075-12 (R2=H). RdmC is obtained by amplifying the rdmC gene by PCR (polymerase chain reaction) technique, using pJN028 as a template and by inserting the fragment amplified into pIJ486 to give pRdm52. R2 is converted to a hydroxyl group by the concomitant action of RdmC and RdmB, wherein the gene product of rdmC is responsible for removing of the carbomethoxy group, while rdmB encodes a hydroxylase causing R2 to be a hydroxyl group. rdmC and rdmB are cloned together to cause the conversion of a carbomethoxy group to a hydroxyl group.
11-hydroxyl group (R3) could be added by the action of the rdmE gene product, a hydroxylase. The gene rdmE is obtained from pJN018 and cloned into pIJE48 to give pRdm51. The suitably fused genes cloned in the expression vector, pIJE486, cause the modifications based on the separate activities of the gene products to generate the derivatives of the compounds characteristic to the mutant strain H075 according to the present invention. Expression constructs were preferably made in S. lividans TK24 to generate large amounts of the recombinant plasmids to be introduced into S. galilaeus H039 exhibiting higher transformation frequency than the other S. galilaeus strains used. The plasmids carrying the combinations of said genes are further isolated from H039 to be introduced into S galilaeus H075 in order to produce compounds of the general formula II above. The relevant plasmids and genes are given in Table 2.
Anthracyclines representing the general formula (II) are produced by fermentation of the H075 clones, i.e. the strains carrying the plasmids listed in Table 2. The strains are H075-pJN028, H075-pSY21, H075-pSYR1, H075-pSYR2, H075-pSYR3, H075-pSYR4, H075-pSYR5, H075-pRdm51, H075-pRdm52, H075-pRdm53 and H075-pRdm54. The anthracyclines produced are isolated by extracting with organic solvents preferably using dichloromethane:methanol solution. The anthracycline of interest is separated from the mixture by column chromatography in two stages. The cytostatic activities of the purified compounds are studied using tumor cells in vitro, and in leukemia model in vivo.
In the following the detailed embodiments of the invention are described as examples of (i) carrying out the mutagenization of the S. galilaeus wild type (ATCC 31615), (ii) construction of the plasmids to generate the modifications for the aglycone, (iii) introduction of the plasmids into S. galilaeus H075 to obtain the H075 clones, (iv) isolation of the compounds produced by H075 clones, purification and identification procedures for the said compounds representing each modification caused by the genes cloned and (v) determination of cytotoxic activities in vitro and in vivo.